Adoptive T-cell therapy is a promising treatment strategy for cancers resistant to conventional methods including surgery, chemotherapy, and radiation therapy. In particular, the adoptive transfer of T cells genetically modified to express tumor-targeting chimeric antigen receptors (CARs) has shown clinical efficacy by redirecting T-cell specificity toward indolent tumors. However, important challenges remain in the use of CAR-modified T cells, including off-target toxicity toward normal cells and susceptibility to mutational escape by targeted tumors. The goal of this research is to improve the safety and efficacy of adoptive T-cell therapy by engineering more robust and versatile tumor-targeting T cells, which will be achieved through two specific aims. In Specific Aim 1, next-generation CARs capable of logical computation of multiple input signals will be developed. OR-gate CARs that trigger T-cell-mediated cytotoxicity in response to multiple tumor-associated antigens will be developed to lower the probability of mutational escape (i.e., loss of all targeted antigens) by tumor cells. AND- and NOT-gate CARs that trigger cytotoxicity only in the presence of the correct combination of antigens will be constructed to lower off-target toxicity toward normal cells. In Specific Aim 2, inducible transcription systems responsive to tumor-specific environmental cues-including hypoxia and increased local concentrations of the immunosuppressive cytokine transforming growth factor beta (TGF- )-will be constructed to express gene products that enhance anti-tumor immune responses, including increased T-cell proliferation and the recruitment of native immune system components to tumor sites. These inducible transcription systems will be combined with the logic-gate CARs developed in Specific Aim 1 to generate tumor-targeting T cells capable of both executing and recruiting robust anti-tumor responses to diseased targets. The proposed receptors and transcription systems will be constructed using rapid, modular DNA assembly technologies developed in the field of synthetic biology. The novel genetic constructs will be stably integrated into established and primary human T cells via lentiviral transduction to enable performance characterization and system optimization. In vitro assays including western blots, surface and intracellular antibody staining, cytokine production profiling, and chromium release (cell lysis) assays will be performed to ascertain the expression and functional activities of new receptors and transcription systems. Constructs showing robust in vitro performance will be further examined in tumor xenograft models in mice to evaluate their effects on tumor eradication by modified T cells. This research aims to address a critical barrier to progress in T-cell therapy for cancer by pursuing the de novo construction of multi-functional genetic constructs previously unavailable in the T-cell therapy toolbox, thereby generating T cells with more robust and precisely targeted anti-tumor activities for immunotherapy against cancer. PUBLIC HEALTH RELEVANCE: Adoptive T-cell therapy for cancer, or the use of tumor-targeting T cells derived or modified from cancer patients' own blood cells, has shown effectiveness in treating diseases such as metastatic melanoma and B- cell leukemia that are resistant to conventional therapies7, 8. However, adoptive T-cell therapy has been limited to experimental trials thus far due to imperfections in tumor-targeting specificity and a general lack of control over the fate and function of engineered T cells after introduction into patients. The proposed research aims to improve the safety and efficacy of T-cell therapy by engineering T cells with more precise and robust tumor targeting and eradication capabilities, thus contributing to the advancement of a new therapeutic strategy against currently incurable diseases.